This invention relates to enzyme electrodes; in particular it relates to a method for controlling the enzyme activity in an enzyme electrode useful for analytical purposes, and more particularly it relates to a method for controlling substrate specific multisubstrate enzymes utilizing a thin-layer electrochemical cell to control the activity of the enzyme.
Polarographic cell systems have become quite popular in recent years for measurement of various substances. In addition, enzymes have been used in polarographic cells, especially in instances where the unknown substance to be measured is not itself polarographically active, but a material produced or consumed by an enzymatic reaction with that unknown is detectable. For example, it is known that galactose is not polarographically active but that the following reaction takes place in the presence of the enzyme galactose oxidase: EQU galactose+O.sub.2 .fwdarw.H.sub.2 O.sub.2 +galactohexodialdose
The hydrogen peroxide produced by the reaction can be measured in a polarographic cell such as the system taught by Clark, U.S. Pat. No. 3,539,455. Since the hydrogen peroxide produced is in direct proportion to the amount of galactose present, it is theoretically possible to quatitatively determine the amount of glactose present in a sample where this is unknown. Likewise, it is possible to quantitatively determine the amount of galactose present by measuring the amount of oxygen used in the above reaction mechanism.
Unfortunately, the enzyme galactose oxidase is a nonspecific enzyme which catalyzes the production of hydrogen peroxide and oxygen consumption from a variety of substrates including galactose, glycerin, dihydroxyacetone, and glyceraldehyde. The term "substrate" is understood to include a distinct chemical entity or a class of chemical entities. In many instances, two or more of these compounds will be present together. For example, galactose and glycerin are both found in blood plasma. Present polarographic measuring systems using galactose oxidase are incapable of distinguishing between these compounds because of the nonspecificity of galactose oxidase. This is also true of other multisubstrate enzymes. Accordingly, the need exists in the art for a method of controlling the relative substrate preference of multisubstrate enzymes in order to enable determinative polarographic measurements to be made.
It is known that certain enzymes and other proteins show an activity dependence based upon the reduction-oxidation (redox) potentials of solutions containing such enzymes. For example, Santhanam et al, 99 J. American Chemical Society, 274 (1977), reported that the enzyme urease when adsorbed onto the surface of a mercury coated thermistor, reversibly lost activity (as measured by a temperature change of the thermistor) at a given reducing potential. However, this technique has only limited utility for those proteins which will adsorb directly onto mercury, has a slow response time, and is not very sensitive.
Hamilton et al, 1 Oxidases and Related Redox Systems, 103 (1965), in theory teach "control" of the potential of a solution which also contained the enzyme glactose oxidase. Hamilton and his coworkers used a given ratio of ferricyanide to ferrocyanide to chemically control the solution potential. Then, by adding galactose and monitoring the uptake of oxygen with a Clark oxygen electrode to determine activity, they plotted the activity dependence on the solution potential (ratio of ferri-to-ferrocyanide). This approach was time consuming since several solutions had to be made up but, also another problem with it was the uncertainty in the true solution potential seen by the enzyme. This results from the fact that the ratio of ferricyanide to ferrocyanide is not controlled after these compounds are added to the solution and obviously this ratio can change both before and/or during the determination of activity.
Finally, Heineman et al, 47 Anal. Chem. 79 (1975), calculated the formal oxidation-reduction potentials (E.degree..sup.') for several enzymes using a thin layer electrochemical cell. By applying a series of differing potentials to a solution containing the enzyme of interest, the ratio of oxidized to reduced components was measured spectrophotometrically and used to plot a linear graph, the intercept of which yielded a formal redox potential value (E.degree..sup.').
Likewise, Caja in "Thin-Layer Cell for Routine Applications," 61 Analytical Chemistry, 1328 (July 1979), describes a thin layer cell and a wire thin layer electrode. The thin layer electrode was surrounded by Nafion cation exchange tubing. These workers stressed the permselectivity of the cation exchange membrane and the resulting benefit that only small amounts of solutions containing electroactive anions and/or electroactive large neutral species were required for electrochemical studies. No provisions were made for the introduction of substrates under controlled conditions into the thin layer cell. Also, the configuration described would preclude the rapid determination of enzymatic activity due to the slow equilibration of substrate across the thick Nafion membrane.
Thus, to my knowledge no one has utilized the control of redox potential of a solution containing a multisubstrate enzyme to advantage in a polarographic system.
Control of enzyme reaction rates in areas other than polarography has been suggested. Fresnel in U.S. Pat. Nos. 4,016,044 and 3,919,052 does so in the field of manufacture and treatment of food products by enzyme catalysis. Fresnel teaches that regulation of the enzymatic reactions is achieved by applying a potential to an enzymatic electrode (an enzyme fixed on a solid electronically conductive support) and controlling the value of the potential during the reaction so as to compensate for variations in the reaction conditions and the enzyme activity and thereby ensure a constant reaction rate. The system described by Fresnel in the examples in his patents actually performs a maintenance function in the so-called "regulation" of enzymatic activity. The mechanism by which this maintenance of activity is obtained is indirect, evidently by control of environmental factors such as pH, dielectric, etc. which, in turn, can affect enzyme activity. In the '052 patent Fresnel even suggests that the technique "may allow the specificity of--[the] enzyme to be modified, if need be." However, there is nothing disclosed in this patent concerning specificity beyond that broad suggestion.
Accordingly, the need still remains for a method of directly controlling the relative activity of an enzyme, for example controlling the substrate preference of a non-specific enzyme such as galactose oxidase, in order to enable rapid determinative analytical measurements to be made.